EP0596881B1 - Expression dans des lignees lymphoblastoides humaines non-tumorales avec un vecteur integratif - Google Patents

Expression dans des lignees lymphoblastoides humaines non-tumorales avec un vecteur integratif Download PDF

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EP0596881B1
EP0596881B1 EP91914319A EP91914319A EP0596881B1 EP 0596881 B1 EP0596881 B1 EP 0596881B1 EP 91914319 A EP91914319 A EP 91914319A EP 91914319 A EP91914319 A EP 91914319A EP 0596881 B1 EP0596881 B1 EP 0596881B1
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dna
vector plasmid
heterologous
expression cassette
epo
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EP0596881A1 (fr
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Annick Ballay
Georges Boffa
Jean-Pierre Cartron
Stany Chretien
Patrick Lambin
Claude Lopez
Sylvie Prigent
Charles Salmon
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FONDATION NATIONALE DE TRANSFUSION SANGUINE
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C07K14/505—Erythropoietin [EPO]
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    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron

Definitions

  • the invention relates to the use of human cell lines for the production, on an industrial scale, of heterologous proteins, more particularly proteins of therapeutic interest, after genetic manipulation of their coding sequence.
  • the invention relates to the use of human lymphoblastoid cells, immortalized in vitro by the Epstein-Barr virus and selected for their absence of tumor or tumorigenic character.
  • the coding sequence of these proteins is inserted into a genetically manipulated vector plasmid, of integrative type and carrying an expression cassette designed to allow the expression of said proteins in transformed lymphoblastoid cells. , cloned and selected, and having integrated the expression cassette into cellular chromosomal DNA.
  • the invention also relates to the design and construction of this vector plasmid intended to ensure optimal production of heterologous proteins by the transformed lymphoblastoid cells.
  • a human lypmphoblastoid line the Namalwa line, has already been used to produce a heterologous protein; it multiplies well in suspension and secretes the heterologous protein in the medium where it can be recovered, in biologically active form (Yanagi et al. Gene 1989,76,19 and DNA 1989,8,419) but this line is derived from a lymphoma from Burkitt and has a high degree of tumorigenicity.
  • the objective of the present invention is the use of human lymphoblastoid cells not tumor and non-tumorigenic.
  • Such cells are known: by transformation with the Epstein-Barr virus (EBV), it is possible to immortalize B lymphocytes from the blood of any healthy donor, and to select lines which only synthesize the nuclear antigen EBNA-1 of EBV (Thoda et al. Cancer Res. 38, 1978, 3560). Such lines proliferate indefinitely in culture but are not tumor, which is verified by their lack of capacity to form colonies in agar, and they are not tumorigenic on the "nu-nu” mouse (Gurtsevitch et al , Int J. Cancer 47 , 87, 1988, Tursz, Medecine / Sciences 6, Suppl. 42, 1989).
  • EBV Epstein-Barr virus
  • This type of cell has already served as a model for expressing heterologous proteins whose gene is inserted in an autonomous replicon which is maintained in an episomal state in the cell because it carries the origin of replication of the EBV which found under the control of the EBNA-1 protein of EBV which served to immortalize the cell line (Kioussis et al. EMBO J.6, 1987, 355; Young et al. Gene 62, 1988, 171; Jalanko et al Gene 78, 1989, 287).
  • the episomal state of the replicon does not guarantee the stability of its number of copies per cell and therefore the production capacity of the heterologous protein over time, especially on an industrial scale which involves a very large number of cycles. cell multiplication.
  • the present invention also relates to the development, by genetic manipulation, of a vector comprising all the elements necessary for the expression of a heterologous protein in human lymphoblastoid cells but also DNA elements promoting the integration of this set into DNA chromosome of the receptor cell.
  • the present invention relates to the use of human lymphoblastoid cell lines, immortalized in vitro by EBV and selected for their absence of tumor or tumorigenic character, said cells being able to be derived from B lymphocytes of any healthy donor or, among such lines, one can choose a particular and well characterized line, deposited at the ATCC under the reference CCL 156 RPMI 1788.
  • the use according to the invention allows the production, on an industrial scale, of heterologous proteins encoded by a DNA segment adapted to be inserted into a genetically manipulated vector plasmid, of integrative type and carrying an expression cassette comprising all the elements allowing the expression of said heterologous proteins after integration of said expression cassette into the chromosomal DNA of lymphoblastoid cells.
  • the central zone of the expression cassette, intended for the insertion of the heterologous DNA sequence has been designed to offer rare restriction sites to favor this insertion manipulation, and more particularly, the Not I, Bst sites. E II, Bgl II, Mlu I.
  • the invention is more particularly intended for the use of the cells described, with the vector plasmid described, for producing proteins of therapeutic interest which are difficult to purify from natural sources.
  • the invention therefore comprises the insertion of the DNAs coding for these heterologous proteins into the expression cassette as described above.
  • the vector plasmid is then introduced into the cells by electroporation (Potter et al. Proc. Natl. Acad. Sci. USA 81, 7161, 1984).
  • Natural protein is produced in the kidneys and circulates in very small quantities in the plasma. She plays an essential role in the maturation of erythrocytes.
  • IL-3 interleukin 3
  • IL-3 is a 20-26 kDa glycoprotein stimulating the proliferation and differentiation of hematopoietic progenitors belonging to the erythroid, megakaryocytic, granulo-macrophagic, eosinophilic and basophilic lines, whose human cDNA was cloned by Yang et al ( Cell 47,3-10,1986).
  • IL-3 is involved in self-renewal and differentiation of the multipotent stem cell (CFU-S), in synergy with IL-1.
  • CFU-S multipotent stem cell
  • IL-3 also has, in synergy with the specific cytokines of each line, an activity on the growth and the functions of differentiated cells (macrophages and mast cells, neutrophils and eosinophils). Because of its role in stimulating hematopoietic stem cells, IL-3 could find a therapeutic application in the treatment of spinal cord aplasia (Shrader, Annu. Rev. Immunol. 4, 205-230, 1986; Mizel, FASEB J 3, 2379, 1989; Tigaud et al., Medicine / science 7, 444, 1991). IL-3 has also been shown to stimulate hematopoiesis in primates, in synergy with GM-CSF (Donahue et al., Science 241, 1820, 1988).
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • GM-CSF is also a multipoietin, that is to say a growth factor capable, like IL-3, of stimulating the growth and the differentiation of several hematopoietic lines (granulo-monocytic, erythroid and mixed colonies) and act on the functions of differentiated cells (macrophages).
  • the human GM-CSF gene has been cloned by several teams (Wong et al, Science 228, 810, 1985; Lee et al, Proc. Natl. Acad. Sci. USA 82, 4360, 1985; Cantrell et al, Proc.
  • the present invention also covers cell lines after their stable transformation by a vector plasmid as described and which have been cloned and selected by screening, from among many transformed clones, for their high rate of secretion of the chosen heterologous protein.
  • the harvesting and purification processes are adapted to each protein.
  • a genomic DNA library was screened with a 30-mer oligonucleotide: 5'-CGTGATATTCTCGGCCTCCTTGGCCTCCAA-3 'deduced from the gene sequence (Lin et al, Proc. Natl. Acad. Sci. USA 82 , 7580, 1985).
  • This probe is an inverted complementary to the messenger RNA and recognizes the sequence of Epo at position 129-159 (base No 1 representing the initiation of translation).
  • the DNA of these 3 phages was amplified, then analyzed.
  • the restriction map of these 3 clones was constructed and the presence of the 3 ′ and 5 ′ ends of the Epo gene was sought using two oligonucleotide probes recognizing the limits of this gene. .
  • Two of the three clones contained the entire Epo gene. Restriction fragments containing the entire Epo gene were subcloned into an amplification vector of the pUC type. Thus the two restriction fragments Bam HI- Hind III of 5.4 kb and Bgl II- Bgl II of 4 kb were subcloned.
  • RNAs from a human renal tumor constitutively expressing Epo were isolated. From these total RNAs, the poly A + RNA fraction was isolated by passage through an oligo-dT column and cloned by the PCR method.
  • This oligonucleotide (20-mer) is in position 694 with respect to ATG adenosine 1 (initiation of translation of the Epo messenger). This oligonucleotide recognizes the complementary reverse sequence of the Epo mRNA and bounds the 3 'part of the cDNA. This oligonucleotide (20-mer) is in the same direction as the mRNA and bounds the 5 ′ part of the cDNA of Epo (position-10 relative to adenosine 1 of the initiation codon). The INTS 10 probe makes it possible to copy the Epo mRNA matrix using reverse transcriptase.
  • the mRNA is then degraded with sodium hydroxide for 1 hour at 65 ° C.
  • This single-stranded cDNA can then be amplified by the PCR technique using the two probes limiting the cDNA: INTS 10 and INTS 9.
  • a DNA fragment of 723 bp was thus amplified and then isolated by electrophoresis, cloned into a vector. amplification of the pUC type and then sequenced.
  • Epo The coding sequence of Epo (genomic or cDNA) is introduced into the oligonucleotide (L 2 ) of the plasmid pTS39 in the form of a BstE II- Bgl II fragment between the Hind III and Bgl II sites.
  • the cells are then cultivated in a selective medium (Iscove, 10% FCS - fetal calf serum - dialysed, mycophenolic acid 1 ⁇ g / ml, xanthine 250 ⁇ g / ml corresponding to the selection gene XGPRT).
  • a selective medium Iscove, 10% FCS - fetal calf serum - dialysed, mycophenolic acid 1 ⁇ g / ml, xanthine 250 ⁇ g / ml corresponding to the selection gene XGPRT).
  • cloning by limiting dilution is carried out in 96-well microplates in the presence of feeder cells (total human lymphocytes, irradiated at 4000 rds, from healthy donors).
  • CCL156 cells are transfected by electroporation with 30 ⁇ g of plasmid DNA and then subjected 48 h after transfection, in the selective medium containing xanthine and mycophenolic acid.
  • the resistant population is cloned by limiting dilution in the presence of feeder cells (irradiated human lymphocytes).
  • feeder cells irradiated human lymphocytes.
  • clone 156 0.5AF8 secretes approximately 100 to 150 units of Epo per ml of culture medium / 72 h and was followed long term in presence and absence of selection pressure. The results indicate that this clone is stable over 48 passages, ie approximately 7 months of culture in the absence of selection pressure.
  • Southern blot analysis of the total DNA extracted from clone 156 0.5AF8 indicates that the vector pTS39-Epo is integrated in tandem into the cell genome at a rate of 15 to 20 copies per cell, whether the clone is cultured in the presence or in the absence of selection pressure.
  • the 0.5AF8 clone secretes 80 to 100 U Epo / ml in three days of culture in a medium poor in proteins CK4 or CK4N (base medium: Iscove-Ham F12 mixture (1: 1); CK4: supplements: human albumin 50 mg / l, human transferrin (saturated at 30% by addition of FeC13) 1 mg / l, bovine insulin 3 mg / l, linoleic acid 1 mg / l, putrescine 10 ⁇ M, ethanolamine 20 ⁇ M; CK4N, idem plus ribonucleotides: Adenosine 100 mg / l, Cytidine 100 mg / l, Guanosine 100 mg / l, Uridine 100 mg / l). This rate of secretion can be renewed 3 times if the medium is changed every 3 days.
  • Epo activity is measured in vitro based on the incorporation of 59 Fe by mouse fetal liver erythroblasts (taken on the 13th day of gestation) cultured in 26 hours according to a method derived from that described by Stephenson et al, (Endocrinology 88 , 1519, 1971).
  • the cells are mechanically dissociated and suspended in RPMI 1640 medium (Tambourin et al, Biomedicine 19 , 112, 1983; Goldwasser et al, Endocrinology 97 , 315, 1975) at a concentration of 1.6 x 10 6 cells / ml containing 7 % fetal calf serum, 85 ⁇ M albumin and 0.4 ⁇ M human transferrin.
  • a serum-free medium in which purified or synthetic natural phospholipids (soy, ovolecithin) (32 x 10 -3 M) and cholesterol (1.6 x 10 -3 M) are solubilized in chloroform, secondarily evaporated under a stream of nitrogen.
  • the lipids in albumin solution at 1 mg / ml in RPMI are dispersed by ultrasound in melting ice (Boffa et al, Exp. Hematol. 10 , 675, 1982).
  • the cell suspension is distributed in volume of 200 ⁇ l in the round bottom wells of the "Nunclon" M culture plates. After an incubation of 21 hours at 37 ° C in the presence of 0.7% CO 2 , the 59 Fe-transferrin is incorporated into the wells, the plates are shaken and returned to the incubator for a prolonged incubation of 5 hours.
  • Each sample is analyzed at 3 or 4 protein concentrations.
  • the activity of each dose results from a quadruple determination.
  • the rate of incorporation of 59 Fe into the heme extracted with methyl ethyl ketone or into the cells is measured as a function of the logarithm of the dose of the sample and the results are expressed in milliunits / ml or per mg of protein.
  • the standard is recombinant human Epo titrating 100,000 U / mg. Its specific activity was defined according to samples of human Epo from the second preparation of International Reference (Annable et al, Bull. Wld. Hlth. Org. 47 , 99, 1972).
  • Epo HR The effects of Epo HR on the maturation of BFUe and CFUe were determined in culture of erythroid cell precursors on methylcellulose in IMDM serum-free medium (Cormier et al, Cell Differentiation, 17 , 261, 1985). Epo HR, like human urinary Epo, determines the differentiation and maturation of BFUe from mouse bone marrow or human peripheral blood. The dose effect of Epo HR on the development of CFUe is compared with that given by natural Epo.
  • the distribution successively concerns the Epo of the range or the sample in a volume of 100 ⁇ l, the immune serum anti Epo 10 ⁇ l, 125-I-Epo HR 10 ⁇ l and the Tachisorb R 100 ⁇ l. After transfer of the glass fiber samples to the polystyrene tube, the radioactivity is measured.
  • the measurement range of the method is between 1 and 100mU.
  • the supernatants of the transformed cells have activities of up to 800 U / ml, while the cell supernatants with antisense vector are devoid of activity. There is always an excellent correlation between in vitro biological assays and radioimmunoassay.
  • Epo produced in the culture supernatants of transformed lymphoblastoid cells is tested after injection into mice made polyglobular by transfusion in order to inhibit the synthesis of endogenous Epo (Jacobson. Et al Proc. Soc. Exp Biol. Med, 94, 243, 1957).
  • the results indicate that the Epo produced by lymphoblastoid cells has a powerful biological activity comparable to that of Epo HR produced by CHO cells (Kirin-Amgen Patent EP 0 148 605).
  • the cell culture supernatant in a protein-poor medium is concentrated by ultrafiltration on an Amicon PM10 membrane or by tangential filtration on Minitan M equipped with a PLGC membrane followed by washing in 12.5 mM glucose solution, galactose 12, 5 mM, PEG 6000 0.1 mg / ml ⁇ -mercaptoethanol 10 -4 M, up to a resistivity of filtrate greater than 5000 Ohms.
  • the lyophilized concentrate is chromatographed on Ultrogel M AcA44 in 10 mM calcium acetate buffer, 100 mM NaCl, 5.6 10 -4 M phenol, 12.5 mM glucose, 12.5 mM galactose, PEG 6000 0.1 mg / ml pH 6.8.
  • the active fractions are concentrated by ultrafiltration and lyophilized.
  • the product is then treated by HPLC on a TSK 545 DEAE column in 0.048 mM acetate buffer, 3 mM CaCl 2 , 12.5 mM glucose, 12.5 mM galactose, PEG-6000 0.1 mg / ml, ⁇ -mercaptoethanol 10 -4 M then elution by a discontinuous molarity gradient of NaCl.
  • the activity yield is close to 75-%.
  • the product is chromatographed on CM Affigel blue M in 10 mM phosphate buffer, 150 mM NaCl pH 7.2.
  • the Epo is eluted with a molarity of 1.15 M NaCl.
  • the specific activity of Epo HR is greater than 100,000 U / mg.
  • the concentrate of the culture media obtained on ultrafiltration membranes is treated on DEAE Sephacel M at 4 ° C. in 48 mM acetate buffer, 3 mM CaCl 2 , 5.4 10 -4 M phenol, pH 4.5.
  • the selected activity is eluted in 0.6 M NaCl.
  • the eluate is subjected to filtration on Ultrogel M AcA44, then the active fraction detected by RIA is chromatographed by HPLC-DEAE in the presence of stabilizers and hexoses.
  • the Epo HR eluted in pH 5.5 buffer, with low NaCl molarity, is concentrated on an Amicon M PM10 membrane and then loaded onto a column of WGA-Sepharose 6MB in PBS buffer at 4 ° C. Its elution is obtained by N-acetylglucosamine 0.5 M.
  • an additional purification step is carried out either on CM Affigel blue M , or on HA-Ultrogel M or in HPLC filtration in 0.1% SDS medium.
  • the apparent molecular weight of the Epo HR in polyacrylamide / SDS gel is 34 kDa.
  • the specific activity obtained is greater than 120,000 U / mg in culture of erythroblasts and in RIA with yields of between 20 and 30%.
  • Epo HR Epo HR was carried out according to the method described in the second example above.
  • Each mouse received 15 days apart three intraperitoneal injections at the following doses: 90, 45 and 10 or 35 ⁇ g for the last injection. All the fusions were performed 5 days after the last injection.
  • the purification of the antibodies is carried out on protein G-Sepharose.
  • the purified antibody is obtained by adsorption of ascites in 0.1 M phosphate buffer, pH 7.5, washing at pH 6.5 and elution in 0.1 M glycine-HCl buffer, pH 2.8.
  • Lots of 30 mg of purified Ep10-141A26 antibody are obtained and fixed on Sepharose 4B activity (8 ml of gel) with cyanogen bromide.
  • Sepharose 4B activity 8 ml of gel
  • such a column can fix approximately 5 to 6 mg of Epo HR.
  • Epo is adsorbed by the immobilized antibody at a pH close to neutral. Its elution was analyzed at different pH: 6.5, 4.5, 2.8 and 2.2; it is mainly carried out from pH 4.5 to 2.8.
  • the immunoadsorbent is stored in phosphate buffer pH 7.5, Thymirosal M 0.02% at 4 ° C; the column can be used for many cycles.
  • the degree of homogeneity of the Epo HR preparations is assessed by densitometry after electrophoresis in SDS polyacrylamide gel.
  • the Epo is more than 98% homogeneous.
  • the apparent molecular weight measured by SDS polyacrylamide gel electrophoresis is 34,000 daltons. Its electrophoretic behavior is identical to that of Epo produced by CHO cells as well as that of the radioiodinated molecule (marketed by Amersham). Its isoelectric point is between 3.5 and 4. Its specific activity is greater than that of urinary Epo preparations and is, depending on the batch, around 200,000 IU / mg.
  • Example 4 Use of the integrative vector plasmid to produce human Interleukin-3 in lymphoblastoid cells.
  • a probe consisting of the reverse complementary oligonucleotide (30-mer) of an IL-3 sequence was used to screen a genomic DNA library of human leukocytes and a human fetal liver cDNA library.
  • the synthesized probe corresponds to base 110 (and following) downstream from the ATG initiator of the IL-3 cDNA coding sequence.
  • the amplification product of 2.140 bp is subcloned into the Sma I site of a pUC18 and partially sequenced to verify the integrity of the IL-3 gene (according to Yang and Clark, in "Developmental Control of globin gene expression” AR Liss p.3-11 1987).
  • the coding sequence thus controlled was introduced into the expression plasmid of the vector plasmid described in Example 1. A transformed cell clone giving a very positive response was selected and multiplied.
  • Biologically active interleukin-3 is secreted into the cell culture medium.
  • Example 5 Use of the integer vector plasmid to produce human GM-CSF in lymphoblastoid cells.
  • a probe made up of the oligonucleotide inverse complementary (30 mer) of a GM-CSF sequence was used to screen a genomic DNA library of human leukocytes and a human fetal liver cDNA library.
  • the synthesized probe corresponds to base 157 (and following) downstream of the ATG initiator of the coding sequence of GM-CSF.
  • phages containing these genes are subcloned into pUC18 in the form of a Hind III / Eco RI fragment of 3.2 Kb. Before introducing this fragment into the expression plasmid of the vector plasmid, the promoter and 3 'untranslated sequences are eliminated. For this, the region containing the coding sequence is amplified by the PCR method using the following 2 synthetic oligonucleotides: start: position-31 of the ATG (Met initiator) start: position 177 downstream of the stop codon using as a template the genomic clones in pUC18, characterized previously.
  • the 2,240 bp amplification product is subcloned into the Sma I site of a pUC18 and partially sequenced to verify the integrity of the GM-CSF gene (according to Miyatake et al. EMBO J.4, 2561, 1985).
  • the coding sequence thus controlled was introduced into the expression plasmid of the vector plasmid described in Example 1. A transformed cell clone giving a very positive response was selected and multiplied.
  • Biologically active GM-CSF is secreted into the cell culture medium.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
EP91914319A 1991-08-01 1991-08-01 Expression dans des lignees lymphoblastoides humaines non-tumorales avec un vecteur integratif Expired - Lifetime EP0596881B1 (fr)

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PCT/FR1991/000636 WO1993003163A1 (fr) 1991-08-01 1991-08-01 Expression dans des lignees lymphoblastoides humaines non-tumorales avec un vecteur integratif

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EP0596881A1 EP0596881A1 (fr) 1994-05-18
EP0596881B1 true EP0596881B1 (fr) 1997-03-19

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JP (1) JPH06508982A (ja)
AT (1) ATE150486T1 (ja)
DE (1) DE69125292T2 (ja)
DK (1) DK0596881T3 (ja)
ES (1) ES2100953T3 (ja)
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WO1996009399A2 (en) * 1994-09-23 1996-03-28 Somatix Therapy Corporation Chimeric adenovirus for gene delivery
DK2163640T3 (da) * 1999-04-15 2012-03-05 Crucell Holland Bv Rekombinant proteinproduktion i en human celle indeholdende mindst ét E1-protein af adenovirus
US8236561B2 (en) 1999-04-15 2012-08-07 Crucell Holland B.V. Efficient production of IgA in recombinant mammalian cells
US7297680B2 (en) 1999-04-15 2007-11-20 Crucell Holland B.V. Compositions of erythropoietin isoforms comprising Lewis-X structures and high sialic acid content
US6855544B1 (en) 1999-04-15 2005-02-15 Crucell Holland B.V. Recombinant protein production in a human cell
US7604960B2 (en) 1999-04-15 2009-10-20 Crucell Holland B.V. Transient protein expression methods
US7192759B1 (en) 1999-11-26 2007-03-20 Crucell Holland B.V. Production of vaccines
US7527961B2 (en) 1999-11-26 2009-05-05 Crucell Holland B.V. Production of vaccines
US7521220B2 (en) 1999-11-26 2009-04-21 Crucell Holland B.V. Production of vaccines
ATE384785T1 (de) 2001-12-07 2008-02-15 Crucell Holland Bv Herstellung von viren, virusisolaten, und impfstoffen
DK1623023T3 (da) 2003-05-09 2009-03-02 Crucell Holland Bv Kulturer af E1-immortaliserede celler og fremgangsmåder til dyrkning deraf til forögelse af produktudbytter derfra

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ATE161882T1 (de) * 1986-12-16 1998-01-15 Gist Brocades Nv Molekulare klonierung und expression von menschlichem il-3

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 103, 1985, Columbus, OH (US); C. SVENSSON et al., p. 205, no. 136210; and G. LUTFALLA et al., p. 117, no. 48859j *
Chemical Abstracts, vol. 103, 1985, Columbus, Ohio, US; abstract No. 48859J, page 117; column 1; vol. 11, No. 3, 1985, pages 223-238; LUTFALLA, G. et al. *
GENE, vol. 76, 1989, Amsterdam (NL); H. YANAGI et al., pp. 19-26 *

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ATE150486T1 (de) 1997-04-15
DE69125292T2 (de) 1997-09-25
EP0596881A1 (fr) 1994-05-18
DK0596881T3 (da) 1997-10-13
JPH06508982A (ja) 1994-10-13
DE69125292D1 (de) 1997-04-24
WO1993003163A1 (fr) 1993-02-18
ES2100953T3 (es) 1997-07-01

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